Method for treating macrophage migration inhibitory factor (mif)-implicated diseases and conditions with iodo pyrimidine derivatives

ABSTRACT

Compounds useful for the inhibition of macrophage migration inhibitory factor (MIF) are provided herein, having the Formula I: 
     
       
         
         
             
             
         
       
     
     wherein A is selected from the group consisting of aromatic or non-aromatic rings, bicyclic rings, polycyclic rings, alkenes or alkynes; B is H, OH, OR, SR, NH 2 , NHR, or alkyl; R is H or alkyl, and X and Y are independently N or CH, but one of X and Y must be N. Also provided are pharmaceutical compositions that contain a Formula I compound and methods for the treatment of MIF-implicated diseases or conditions that include administering a safe and effective amount of a Formula I compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/498,036, filed Mar. 23, 2012, now pending; which itself is a UnitedStates National Stage application of PCT International PatentApplication Serial No. PCT/US2010/050206, filed Sep. 24, 2010; whichitself claims the benefit of U.S. Provisional Patent Application Ser.No. 61/245,481, filed Sep. 24, 2009. The disclosure of each of theseapplications is incorporated herein by reference in its entirety.

The acquisition of migratory and invasive properties by tumor cells is acentral and often fatal step in neoplastic disease progression. Whilenormal, non-transformed cells have strict growth factor and adhesiverequirements for motility, malignant cells have overcome theserequirements through multiple mechanisms including gain of functiononcogene mutations, growth factor receptor overexpression and/orconstitutive deregulation of extracellular matrix degrading enzymes. Notcoincidentally, many solid cancers also possess very low oxygentensions.

Hypoxia can induce macrophage migration inhibitory factor (MIF)expression. It has been demonstrated that MIF expression is increased inpre-malignant, malignant, and metastatic tumors. Breast, prostate,colon, brain, skin, and lung-derived tumors have all been shown tocontain significantly higher levels of MIF message and protein thantheir non-cancerous cell counterparts. MIF expression closely correlateswith tumor aggressiveness and metastatic potential, possibly suggestingan important contribution to disease severity by MIF. MIF has beenindirectly implicated in tumor growth and progression by stimulatingtumor-dependent stromal processes such as neovascularization. Further,MIF has been implicated in macrophage and lymphocyte activation andsurvival and may play a role in inflammatory disorder progression.

Thus, certain aggressive tumors appear to possess an importantfunctional requirement for MIF in maintaining optimal growth andprogression. MIF therefore provides a valuable target for development oftherapeutics for the treatment of cancer. Further, MIF may be importantin the progression of inflammatory disorders. The need exists to developtherapeutic molecules that target MIF and modulate one or morebiological activities of MIF for the treatment of cancers and otherinflammatory disorders.

Moreover, MIF is produced by several different pathogens includingparasitic helminths, spirochetes, and plasmodium. As such, irreversibleinhibitors of MIF such as 4-iodo-6-phenylpyrimidine (4-IPP) and analogsmay be excellent antagonists of parasite-derived MIF. The need exists todevelop therapeutic molecules that target MIF and ameliorate thedisease-causing pathologies associated with these and otherMIF-producing pathogens.

In one embodiment of the invention, a compound or its enantiomeric ordiastereomeric form or a pharmaceutically acceptable salt, prodrug, ormetabolite thereof is provided, said compound having the formula:

wherein: A is selected from the group consisting of: i) substituted orunsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having 0or 1 to 4 heteroatoms selected from the group consisting of N, O, S, andcombinations thereof; ii) substituted or unsubstituted bicyclic ring;iii) substituted or unsubstituted polycyclic rings; and iv) substitutedor unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1to 3 double or triple bonds; B is H, OH, OR, SR, NH₂, NHR, alkyl orsubstituted alkyl or A, but when B is A, A is H or halo; R is H, alkylor substituted alkyl of 2 to 20 carbon atoms; and X and Y areindependently N or CH, but one of X and Y must be N.

In another embodiment, a pharmaceutical composition is provided,comprising (a) an effective amount of a Formula I compound or itsenantiomeric or diastereomeric form or a pharmaceutically acceptablesalt, prodrug, or metabolite thereof, and (b) one or morepharmaceutically acceptable excipients.

In another embodiment, a method for treating a macrophage migrationinhibitory factor (MIF)-implicated disease or condition is provided, themethod comprising administering to a patient in need thereof aneffective amount of a Formula I compound, or its enantiomeric ordiastereomeric form or a pharmaceutically acceptable salt, prodrug, ormetabolite thereof.

These and other objects, features, embodiments, and advantages willbecome apparent to those of ordinary skill in the art from a reading ofthe following detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts MIF liver enzyme inhibition as a percent of control,comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.

FIG. 2 depicts MIF tumor enzyme inhibition as a percent of control,comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.

FIG. 3 depicts a comparison of IC50 values across the tumor cell linesH23, MCF7, MDA-MB-231, H-460, SKOV-3, PC3, DU145, Miapaca, LnCap, Capan1, Capan 2, and CAOV3.

FIG. 4 depicts a comparison of IC50 values across the tumor cell linesDU145, MDA-MB-231, Miapaca, CAOV03, and HUVEC.

FIG. 5 depicts p53 regulation of compounds compared to control (DMSO),4-IPP, and ISO-1 at 10 μM concentration. Compounds tested includedACT-MIF-030, ACT-MIF-035, ACT-MIF-038, ACT-MIF-029, ACT-MIF-033,ACT-MIF-034, ACT-MIF-003, and ACT-MIF-028. Results indicate thecompounds are implicated in p53 regulation.

FIG. 6 depicts inhibition of cell proliferation IC50 values forACT-MIF-006, ACT-MIF-035, and ACT-MIF-038 in the LOX-IMV1 tumor cellline.

FIGS. 7A-7D depict inhibition of cell migration in the LOX-IMV1 tumorcell line at 72 hrs, for ACT-MIF-006 (A), ACT-0035 (B and D), andACT-MIF-038 (C). Results indicate a significant inhibition of migration,even at low concentrations (0.03 μM).

FIG. 8 depicts tumor growth inhibition of DU145 human prostatexenografts in athymic nude mice treated with ACT-MIF-001, ACT-MIF-002,and ACT-MIF-003. Results show ACT-MIF-003 significantly inhibited tumorgrowth.

FIGS. 9A-9D depict tumor slices from animals treated with control (A),ACT-MIF-002 (B), ACT-MIF-001 (C), and ACT-MIF-003 (D). Blood vesseldensity of the tumor tissues was measured by immunohistochemistry.Results indicated a decrease in microvessel density with respect to thetumors of the control group with a statistically meaningful differencefor the ACT-MIF-003 treated group.

FIGS. 10A and 10B depict tumor growth inhibition (A) and survival data(B) in a pancreatic tumor model treated with control, ACT-MIF-002, andACT-MIF-003. Results indicate the tested compounds had significantimpact on survival and limited metastatic tumor burden.

FIGS. 11A and 11B depict histopathological slides comparing bone marrowfrom pancreatic tumor model animals treated with control (A) andACT-MIF-002 (B). Bone marrow of the vehicle treated mice is consistentwith bone metastases (1) with evidence of surrounding skeletal musclemetastases from invading marrow tumor cells (2 and 3). No evidence ofbone metastases was observed with spinal column sections fromACT-MIF-002 treated mice.

FIG. 12 depicts a comparison of MIF enzyme inhibition in the liver,brain, and lung of healthy animals administered ACT-MIF-002 eitherintraperitoneally (IP) or per oral (PO). Results indicate the compoundis orally bioavailable, crosses the brain blood barrier, and inhibitsMIF enzymatic activity in both the brain and the lungs.

FIGS. 13A-13D depict 4-IPP-based MIF antagonists effects on primary Tlymphocyte activation/proliferation. Fresh, primary human T lymphocyteswas collected by aphaeresis and separated by Ficoll gradients. 1×10⁶lymphocytes were added to immobilized anti-CD3 tissue culture plates inthe presence of nothing (control; FIG. 13A), vehicle control (0.1% DMSO;FIG. 13B), 25 μM 4-IPP (FIG. 13C), or 25 μM ACT-003 (FIG. 13D). 48 hourslater cells were collected, washed, and stained with anti-CD4 andant-CD8 labeled antibodies followed by flow cytometric analyses. Shownare the relative percentages of CD4/CD8 lymphocytes.

FIGS. 14A and 14B depict the data from FIG. 15 as an overlay of relativefluorescence intensity of expression of CD4 (FIG. 14A) or CD8 (FIG. 14B)in PBMCs activated with plate bound anti-CD3 for 48 hours.

FIGS. 15A-15D depict 4-IPP-based MIF antagonists effects on primary Tlymphocyte activation/proliferation. Fresh, primary human T lymphocyteswas collected by aphaeresis and separated by Ficoll gradients. 1×10⁶lymphocytes were added to immobilized anti-CD3 tissue culture plates inthe presence of nothing (control; FIG. 15A), vehicle control (0.1% DMSO;FIG. 15B), 25 μM 4-IPP (FIG. 15C), or 25 μM ACT-003 (FIG. 15D). 48 hourslater cells were collected, washed, and stained with ananti-CD25-labeled antibody followed by flow cytometric analyses. CD25(high affinity IL-2 receptor) is a commonly used marker for T lymphocyteactivation. Shown are the relative percentages of CD25+ (i.e.,activated) treated vs. untreated lymphocytes.

FIG. 16 depicts the data from FIG. 17 as an overlay of fluorescenceintensity of expression of CD25 in PBMCs activated with plate boundanti-CD3 for 48 hours.

FIGS. 17A-17D depict 4-IPP-based MIF antagonists effects on primary Tlymphocyte activation/proliferation. Fresh, primary human T lymphocyteswas collected by aphaeresis and separated by Ficoll gradients. 1×10⁶lymphocytes were added to immobilized anti-CD3 tissue culture plates inthe presence of nothing (control; FIG. 17A), vehicle control (0.1% DMSO;FIG. 17B), 25 μM 4-IPP (FIG. 17C), or 25 μM ACT-003 (FIG. 17D). 16 hourslater cells were collected, washed, and stained with ananti-CD69-labeled antibody followed by flow cytometry analysis. CD69 isan early marker of lymphocyte activation and the lack of a large effecton early lymphocyte activation suggests that treatment of established Tcell-dependent autoimmune diseases with 4-IPP-based anti-MIF antagonistsis feasible. Shown are the relative percentages of CD69 on treated vs.untreated lymphocytes.

FIG. 18 depicts the data from FIG. 19 as an overlay of fluorescenceintensity of expression of CD69 in PBMCs activated with plate boundanti-CD3 for 48 hours.

FIGS. 19A-19D depict 4-IPP-based MIF antagonists' effects on primary Tlymphocyte activation/proliferation. Fresh, primary human T lymphocyteswas collected by aphaeresis and separated by Ficoll gradients. 1×10⁶lymphocytes were added to immobilized anti-CD3 tissue culture plates inthe presence of nothing (control; FIG. 19A), vehicle control (0.1% DMSO;FIG. 19B), 25 μM 4-IPP (FIG. 19C), or 25 μM ACT-003 (FIG. 19D). 48 hourslater labeled-BrdU was added to the cells briefly, then washed, stainedwith labeled-anti-CD8 antibodies and analyzed for BrdU incorporationinto DNA (readout for proliferation) by flow cytometry.

FIGS. 20A-20D depict 4-IPP-based MIF antagonists' effects on primary Tlymphocyte activation/proliferation. Fresh, primary human T lymphocyteswas collected by aphaeresis and separated by Ficoll gradients. 1×10⁶lymphocytes were added to immobilized anti-CD3 tissue culture plates inthe presence of nothing (control; FIG. 20A), vehicle control (0.1% DMSO;FIG. 20B), 25 μM 4-IPP (FIG. 20C), or 25 μM ACT-003 (FIG. 20D). 48 hourslater labeled-BrdU was added to the cells briefly, then washed, stainedwith labeled-anti-CD4 antibodies and analyzed for BrdU incorporationinto DNA (readout for proliferation) by flow cytometry.

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

The terms “enantiomer” and “diastereomer” have the standard artrecognized meanings (see e.g., Hawley's Condensed Chemical Dictionary,14th ed.). The illustration of specific protected forms and otherderivatives of the compounds of the instant invention is not intended tobe limiting. The application of other useful protecting groups, saltforms, etc. is within the ability of the skilled artisan.

The term “prodrug” refers to any covalently bonded carriers whichrelease the active parent drug according to the Formula I describedabove in vivo when such prodrug is administered to a subject. Prodrugsof the compounds are prepared by modifying functional groups present inthe compounds in such a way that the modifications are cleaved, eitherin routine manipulation or in vivo, to the parent compounds.

The term “substituted” is defined herein as “encompassing moieties orunits which can replace one or more hydrogen atoms of a hydrocarbylmoiety. The term “hydrocarbyl” is defined herein as any organic unit ormoiety which is comprised of carbon atoms and hydrogen atoms.

“Halo” or “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “aromatic ring” refers to an aromatic hydrocarbon ring system.Suitable aromatic rings of embodiments of the present invention contain5, 6, or 7 carbon atoms in the ring. Aromatic rings can also contain 0or 1-4 heteroatoms selected from the group consisting of N, O, S, andcombinations thereof. Non-limiting examples of suitable aromatic ringsinclude phenyl, pyridinyl, pyrimidinyl, pyridazinyl, furanyl,thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, and thiadiazolyl. Aromaticrings of the present invention can be unsubstituted or substituted withfrom 1 to 3 substituents. Non-limiting examples of suitable substituentsinclude halo, hydroxyl, alkoxy, amino, substituted amino, carboxylicacid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl,combinations thereof, or functional equivalents thereof.

The term “non-aromatic ring” refers to a non-aromatic saturated orunsaturated hydrocarbon ring system. Suitable non-aromatic rings ofembodiments of the present invention contain 5, 6, or 7 carbon atoms inthe ring. Non-aromatic rings can also contain 0 or 1-4 heteroatomsselected from the group consisting of N, O, S, and combinations thereof.Non-aromatic rings of the present invention can be unsubstituted orsubstituted with from 1 to 3 substituents. Non-limiting examples ofsuitable substituents include halo, hydroxyl, alkoxy, amino, substitutedamino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof.

The term “bicyclic ring” refers to two fused hydrocarbon rings that mayoptionally include one or more heteroatoms as ring members. A bicyclicring can be substituted or unsubstituted, including single or multiplesubstitutions. The rings can independently show a different degree ofsaturation and may be saturated, unsaturated, or aromatic. Fusion of therings can occur in three ways: across a bond between two atoms; across asequence of atoms (bridgehead); or at a single atom (spirocyclic).Bicyclic rings of the present invention include, but are not limited to,6-5, 6-6, 6-7, 5-5, 5-6, 5-7, 7-5, and 7-6 ring systems, wherein theintegers refer to the number of carbon atoms or heteroatoms in each ringin the structure. Bicylic rings of the present invention can beunsubstituted or substituted with from 1 to 4 substituents. Non-limitingexamples of suitable substituents include halo, hydroxyl, alkoxy, amino,substituted amino, carboxylic acid, ester, amide, substituted amide,nitro, alkyl, substituted alkyl, combinations thereof, or functionalequivalents thereof. Non-limiting examples of suitable bicyclic rings ofthe present invention include indole, quinoline, and naphthalene.

The term “polycyclic ring” refers to three or more fused hydrocarbonrings that may optionally include one or more heteratoms as ringmembers. A polycyclic ring can be substituted or unsubstituted,including single or multiple substitutions. The rings can independentlyshow a different degree of saturation and may be saturated, unsaturated,or aromatic. Fusion of the rings can occur in three ways: across a bondbetween two atoms; across a sequence of atoms (bridgehead); or at asingle atom (spirocyclic). Polycyclic rings of the present invention canbe unsubstituted or substituted with from 1 to 4 substituents.Non-limiting examples of suitable substituents include halo, hydroxyl,alkoxy, amino, substituted amino, carboxylic acid, ester, amide,substituted amide, nitro, alkyl, substituted alkyl, combinationsthereof, or functional equivalents thereof.

The term “alkene” refers herein to a hydrocarbon chain having from 1 to3 carbon-carbon double bonds and having 2 to 10 carbon atoms. Alkenes ofthe present invention can be unsubstituted or substituted with from 1 to3 substituents. Non-limiting examples of suitable substituents includehalo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid,ester, amide, substituted amide, nitro, alkyl, substituted alkyl,combinations thereof, or functional equivalents thereof.

The term “alkyne” refers herein to a hydrocarbon chain having from 1 to3 carbon-carbon triple bonds and having 2 to 10 carbon atoms. Alkynes ofthe present invention can be unsubstituted or substituted with from 1 to3 substituents. Non-limiting examples of suitable substituents includehalo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid,ester, amide, substituted amide, nitro, alkyl, substituted alkyl,combinations thereof, or functional equivalents thereof.

The term “alkyl” refers to a saturated hydrocarbon chain having 2 to 20carbon atoms. Alkyls of the present invention can be substituted orunsubstituted. Non-limiting examples of suitable substituents includehydroxyl, amino, thiol, morpholino, pyrrolidino, piperidino, glycol, andpolyethyleneglycol (PEG) having molecular weight of 200 to 20,000.

The term “pharmaceutically-acceptable excipient,” as used herein, meansany physiologically inert, pharmacologically inactive material known toone skilled in the art, which is compatible with the physical andchemical characteristics of the particular CEL inhibitor selected foruse. Pharmaceutically-acceptable excipients include, but are not limitedto, polymers, resins, plasticizers, fillers, lubricants, diluents,binders, disintegrants, solvents, co-solvents, buffer systems,surfactants, preservatives, sweetening agents, flavoring agents,pharmaceutical grade dyes or pigments, and viscosity agents.

The term “MIF-implicated disease or condition” refers to a disease orcondition for which MIF is a factor in the onset and/or progression ofthe disease or condition.

The term “safe and effective amount” of a Formula (I) compound is anamount that is effective to inhibit the MIF enzyme in an animal,specifically a mammal, more specifically a human subject, without undueadverse side effects (such as toxicity, irritation, or allergicresponse), commensurate with a reasonable benefit/risk ratio when usedin the manner of this invention. The specific “safe and effectiveamount” will, obviously, vary with such factors as the particularcondition being treated, the physical condition of the patient, theduration of treatment, the nature of concurrent therapy (if any), thespecific dosage form to be used, the excipient employed, the solubilityof the Formula (I) compound therein, and the dosage regimen desired forthe composition.

The term “inflammatory disease” refers to a disease characterized byinflammation, or the complex vascular and immune response to harmfulstimuli. Inflammatory diseases include those diseases in whichinflammation and immune cells are involved in the pathology of thedisease. In a specific embodiment, the inflammatory disease is selectedfrom the group consisting of dermatitis, arthritis, rheumatoidarthritis, insulin-dependent diabetes, proliferative vascular disease,acute respiratory distress syndrome, sepsis, septic shock, psoriasis,asthma, cytokine related toxicity, lupus, multiple sclerosis,transplant-host response, and autoimmune disorders.

Compounds according to the present invention have the following genericstructure:

wherein:

-   -   A is selected from the group consisting of:        -   i) substituted or unsubstituted 5, 6 or 7-membered aromatic            or nonaromatic rings having 0 or 1 to 4 heteroatoms selected            from the group consisting of N, O, S, and combinations            thereof;        -   ii) substituted or unsubstituted bicyclic ring;        -   iii) substituted or unsubstituted polycyclic rings; and        -   iv) substituted or unsubstituted alkenes and alkynes having            2 to 10 carbon atoms with 1 to 3 double or triple bonds;    -   B is H, OH, OR, SR, NH₂, NHR, alkyl or substituted alkyl or A,        but when B is A, A is H or halo;    -   R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and    -   X and Y are independently N or CH, but one of X and Y must be N.

In one embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H; and X and Y are both N.

In another embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H, OH, OR, SR, NH₂, NHR, alkyl, or substituted alkyl; Xand Y are both N.

In another embodiment, A is halo, B is selected from the groupconsisting of: substituted or unsubstituted 5, 6 or 7-membered aromaticor nonaromatic rings having none or 1 to 4 heteroatoms which could be asingle atom or the combination of N, O and S; substituted orunsubstituted bicyclic ring, for example indole, quinoline andnaphthalene; substituted or unsubstituted polycyclic rings; andsubstituted or unsubstituted alkenes and alkynes having 2 to 10 carbonatoms with 1 to 3 double or triple bonds; wherein substitutions for anyof the above are selected from the group consisting of halo, hydroxyl,alkoxy, amino, substituted amino, carboxylic acid, ester, amide,substituted amide, nitro, alkyl, substituted alkyl, combinationsthereof, or functional equivalents thereof, and X and Y are both N.

In another embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H; X is N, and Y is CH.

In still another embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H; X is CH; and Y is N.

In another embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H, OH, OR, SR, NH₂, NHR, alkyl or substituted alkyl; X isN and Y is CH.

In still another embodiment, A is selected from the group consisting of:substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromaticrings having none or 1 to 4 heteroatoms which could be a single atom orthe combination of N, O and S; substituted or unsubstituted bicyclicring, for example indole, quinoline and naphthalene; substituted orunsubstituted polycyclic rings; and substituted or unsubstituted alkenesand alkynes having 2 to 10 carbon atoms with 1 to 3 double or triplebonds; wherein substitutions for any of the above are selected from thegroup consisting of halo, hydroxyl, alkoxy, amino, substituted amino,carboxylic acid, ester, amide, substituted amide, nitro, alkyl,substituted alkyl, combinations thereof, or functional equivalentsthereof; B is H, OH, OR, SR, NH₂, NHR, alkyl, or substituted alkyl; X isCH; and Y is N.

In another embodiment, the compound is selected from the group set forthin Table 1.

TABLE 1 EXAM- ACT-MIF PLE NO. CHEMICAL NAME  2 ACT-MIF-4-Iodo-6-(2,3-difluoro-4-methoxyphenyl) 001 pyrimidine  3 ACT-MIF-4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine 002  4 ACT-MIF-4-Iodo-6-(2-fluorophenyl)pyrimidine 003  5 ACT-MIF-4-Iodo-6-(4-fluorophenyl)pyrimidine 004  6 ACT-MIF-4-Iodo-6-(furan-3-yl)pyrimidine 005  7 ACT-MIF-4-Iodo-6-(pyridin-3-yl)pyrimidine 006  8 ACT-MIF-4-Iodo-6-(3-fluorophenyl)pyrimidine 008  9 ACT-MIF-4-Iodo-6-(4-tert-butyloxymethylphenyl) 010 pyrimidine 10 ACT-MIF-4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine 011 11 ACT-MIF-4-Iodo-6-(furan-2-yl)pyrimidine 012 12 ACT-MIF-4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine 013 13 ACT-MIF-4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine 014 14 ACT-MIF-4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine 015 15 ACT-MIF-4-Iodo-6-(2-hydroxyphenyl)pyrimidine 016 16 ACT-MIF-4-Iodo-6-(2,4-difluorophenyl)pyrimidine 017 17 ACT-MIF-4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine 018 18 ACT-MIF-4-Iodo-6-(2-chlorophenyl)pyrimidine 019 19 ACT-MIF-4-Iodo-6-(3-acetylaminophenyl)pyrimidine 021 20 ACT-MIF-4-Iodo-6-(thiophen-3-yl)pyrimidine 022 21 ACT-MIF-4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine 023 22 ACT-MIF-4-Iodo-6-(isoquinolin-4-yl)pyrimidine 025 23 ACT-MIF-4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine 027 24 ACT-MIF-4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine 028 25 ACT-MIF-4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine 029 26 ACT-MIF-4-Iodo-6-(thiophen-2-yl)pyrimidine 030 27 ACT-MIF-4-Iodo-6-(3,4-difluorophenyl)pyrimidine 032 28 ACT-MIF-4-Iodo-6-(4-ethoxyphenyl)pyrimidine 033 29 ACT-MIF-4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine 034 30 ACT-MIF-4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine 035 31 ACT-MIF-4-Iodo-6-(quinolin-4-yl)pyrimidine 036 324-Iodo-6-(quinolin-8-yl)pyrimidine 33 4-Iodo-6-(quinolin-3-yl)pyrimidine34 4-Iodo-6-(isoquinolin-5-yl)pyrimidine 362-Methylthio-4-iodo-6-phenylpyrimidine 372-Ethylthio-4-iodo-6-phenylpyrimidine 382-Isopropylthio-4-iodo-6-phenylpyrimidine 392-n-Butylthio-4-iodo-6-phenylpyrimidine 412-Methylamino-4-iodo-6-phenylpyrimidine 422-Ethylamino-4-iodo-6-phenylpyrimidine 432-Propylamino-4-iodo-6-phenylpyrimidine 442-Isopropylamino-4-iodo-6-phenylpyrimidine 452-n-Butylamino-4-iodo-6-phenylpyrimidine 464-Iodo-6-(benzothiophen-2-yl)pyrimidine 474-Iodo-6-(benzofuran-2-yl)pyrimidine 484-Iodo-6-(4-hydroxybenzothiophen-2- yl)pyrimidine 494-Iodo-6-(4-acetylaminobenzothiophen-2- yl)pyrimidine 504-Iodo-6-(4-aminocarbonylbenzothiophen-2- yl)pyrimidine 514-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine 524-Iodo-6-(5-aminocarbonylpyridin-3- yl)pyrimidine 534-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine 544-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine 554-Iodo-6-(4-aminocarbonylthiophen-2- yl)pyrimidine 564-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine

In another embodiment, X and Y are both N. In another embodiment, when Xand Y are both N, Bis H.

In still another embodiment, A is halo, B is A, and X and Y are both N.In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment,when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, whenX is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting ofindole, quinoline, and naphthalene.

In a very specific embodiment, the compound is4-Iodo-6-(2-fluorophenyl)pyrimidine or4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In another embodiment, a pharmaceutical composition is provided,comprising:

-   -   a) a safe and effective amount of a compound or its enantiomeric        or diastereomeric form or a pharmaceutically acceptable salt,        prodrug, or metabolite thereof, said compound having the        formula:

-   -   -   wherein:        -   A is selected from the group consisting of:            -   i) substituted or unsubstituted 5, 6 or 7-membered                aromatic or nonaromatic rings having 0 or 1 to 4                heteroatoms selected from the group consisting of N, O,                S, and combinations thereof;            -   ii) substituted or unsubstituted bicyclic ring;            -   iii) substituted or unsubstituted polycyclic rings; and            -   iv) substituted or unsubstituted alkenes and alkynes                having 2 to 10 carbon atoms with 1 to 3 double or triple                bonds;        -   B is H, OH, OR, SR, NH₂, NHR, alkyl or substituted alkyl or            A, but when B is A, A is H or halo;        -   R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms;            and        -   X and Y are independently N or CH, but one of X and Y must            be N; and

    -   b) one or more pharmaceutically acceptable excipients.

In one embodiment, the compound is selected from the group set forth inTable 1.

In another embodiment, X and Y are both N. In another embodiment, when Xand Y are both N, Bis H.

In still another embodiment, A is halo, B is A, and X and Y are both N.In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment,when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, whenX is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting ofindole, quinoline, and naphthalene.

In a very specific embodiment, the compound is4-Iodo-6-(2-fluorophenyl)pyrimidine or4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In a further embodiment, a method for treating a macrophage migrationinhibitory factor (MIF)-implicated disease or condition is provided, themethod comprising administering to a patient in need thereof a safe andeffective amount of a compound or its enantiomeric or diastereomericform or a pharmaceutically acceptable salt, prodrug, or metabolitethereof, said compound having the formula:

wherein:

-   -   A is selected from the group consisting of:        -   i) substituted or unsubstituted 5, 6 or 7-membered aromatic            or nonaromatic rings having 0 or 1 to 4 heteroatoms selected            from the group consisting of N, O, S, and combinations            thereof;        -   ii) substituted or unsubstituted bicyclic ring;        -   iii) substituted or unsubstituted polycyclic rings; and        -   iv) substituted or unsubstituted alkenes and alkynes having            2 to 10 carbon atoms with 1 to 3 double or triple bonds;    -   B is H, OH, OR, SR, NH₂, NHR, alkyl or substituted alkyl or A,        but when B is A, A is H or halo;    -   R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and    -   X and Y are independently N or CH, but one of X and Y must be N.

In one embodiment, the compound is selected from the group set forth inTable 1.

In another embodiment, X and Y are both N. In another embodiment, when Xand Y are both N, B is H.

In still another embodiment, A is halo, B is A, and X and Y are both N.In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment,when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, whenX is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting ofindole, quinoline, and naphthalene.

In a very specific embodiment, the compound is4-Iodo-6-(2-fluorophenyl)pyrimidine or4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In one embodiment, the MIF-implicated disease is selected from the groupconsisting of inflammatory disease and cancer.

In a specific embodiment, the inflammatory disease is selected from thegroup consisting of dermatitis, arthritis, rheumatoid arthritis,insulin-dependent diabetes, proliferative vascular disease, acuterespiratory distress syndrome, sepsis, septic shock, psoriasis, asthma,cytokine related toxicity, lupus, multiple sclerosis, transplant-hostresponse, and autoimmune disorders.

MIF is produced by several different pathogens, including parasitichelminths, spirochetes, and plasmodium. Thus, irreversible inhibitors ofMIF, such as the MIF inhibitors of Formula I, are useful as antagonistsof parasite-derived MIF. Accordingly, in a further embodiment, theMIF-implicated condition is caused by a MIF-producing pathogen. In aspecific embodiment, the MIF-producing pathogen is selected from thegroup consisting of parasitic helminths, spirochetes, and plasmodium.

EXAMPLES

These following exemplary embodiments and synthetic schemes are providedby way of illustration only and are in no way intended to limit thescope of the present invention.

Example 1

Methods for the Preparation of 4-Iodo-6-arylpyrimidine Derivatives,where Aryl is Substituted Phenyl, Heterocyclic, or Bicyclic Ring

General Procedure:

4,6-Dichloropyrimidine (1) is reacted with corresponding aryl boronicacid (2) in dioxane- and aqueous sodium carbonate in the presence of acatalyst used for Suzuki coupling at 50 to 100° C. temperature. Theresultant 4-chloro-6-arylpyrimidine (3) is isolated by crystallizationor column chromatography on silica gel and is converted to corresponding4-iodo-6-arylpyrimidine (4) using hydroiodic acid. Further treatment ofHI may be needed when the reaction is not complete.

The compounds of Examples 2-34 are prepared using Scheme 1.

Example 2 4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine(ACT-MIF-001)

The compound was prepared according to EXAMPLE 1 using2,3-difluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.98 (s, 1H), 8.30 (s, 1H), 7.92 (m, 1H), 7.21 (m,1H), 3.98 (s, 3H).

Example 3 4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine (ACT-MIF-002)

The compound was prepared according to EXAMPLE 1 using2-fluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.91 (s, 1H), 8.23 (s, 1H), 8.05 (m, 1H), 7.01 (m,2H), 3.88 (s, 3H).

Example 4 4-Iodo-6-(2-fluorophenyl)pyrimidine (ACT-MIF-003)

The compound was prepared according to EXAMPLE 1. Specifically, thefollowing method was employed:

1. Preparation of 4-chloro-6-(2-fluoro-phenyl)-pyrimidine (3)(TRM/AP/005/127)

4,6-dichloropyrimidine (20.3 g, 136.3 mmol), 2-fluorophenyl boronic acid(20.0 g, 142.9 mmol, 1.05 equiv), Na₂CO₃ (23.4 g, 106.0 mmol, 1.8 equiv)and Pd(PPh₃)₂Cl₂ (1.0 g, 1.4 mmol, 0.01 equiv) were refluxed indimethoxyethane-water (817:272 mL) mixed solvent system for 6.5 h.Reaction was monitored by TLC (using ethyl acetate:n-hexane, 1:9).Reaction mixture was cooled and the subject compound was extracted usingdichloromethane. Subject compound was purified by flash chromatography(2.5% ethyl acetate:n-hexane) to yield 4.5 g (Yield=15.8%).

¹H NMR (CDCl₃): 9.07 (s, 1H), 8.19 (t, J=7.8 Hz, 1H), 7.91 (s, 1H),7.48-7.55 (m, 1H), 7.18-7.35 (m, 2H)

4,6-dichloropyrimidine (5.1 g, 34.1 mmol), 2-fluorophenyl boronic acid(5.0 g, 35.7 mmol, 1.05 equiv), Na₂CO₃ (6.9 g, 65.0 mmol, 1.8 equiv) andPd(PPh₃)₂Cl₂ (0.3 g, 0.4 mmol, 0.01 equiv) were refluxed indimethoxyethane-water (204:69 mL) mixed solvent system for 4 h. Reactionwas monitored by TLC (using ethyl acetate-hexane, 1:9). Reaction mixturewas cooled and the subject compound was extracted using dichloromethane.Subject compound was purified by flash chromatography (2.5% ethylacetate in n-hexane) to yield 3.7 g (Yield=52.0%).

2. Preparation of 4-(2-fluoro-phenyl)-6-iodo-pyrimidine (4)(TRM/AP/006/064)

A solution of 4-chloro-6-(2-fluoro-phenyl)-pyrimidine (7.0 g, 33.6 mmol)in 350 mL acetone was charged with sodium iodide (25.9 g, 172.8 mmol,5.1 equiv) and aqueous solution of HI (241.9 g, 1.9 mol, 56.4 equiv) andstirred continually for 15 h. Reaction mixture was then made slightlyalkaline (pH ˜10) by using 5% NaOH solution. Subject compound wasprecipitated out, filtered, washed well with distilled water and driedunder vacuum to yield 10.0 g of 4 (Yield=99.3%).

¹H NMR (DMSO-d₆): 9.02 (s, 1H), 8.35 (s, 1H), 8.01-8.07 (m, 1H),7.60-7.65 (m, 1H), 7.38-7.44 (m, 2H)

HPLC=98.55%

Example 5 4-Iodo-6-(4-fluorophenyl)pyrimidine (ACT-MIF-004)

The compound was prepared according to EXAMPLE 1 using4-fluorophenylboronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 8.90 (s, 1H), 8.61 (s, 1H), 8.30 (m, 2H), 7.38 (m,2H).

Example 6 4-Iodo-6-(furan-3-yl)pyrimidine (ACT-MIF-005)

The compound was prepared according to EXAMPLE 1 using furan-3-boronicacid and 4,6-dichloropyrimidine. The resultant chloro compound wasconverted to iodo with hydroiodic acid as described in the generalprocedure.

¹H NMR (DMSO-d₆): δ 8.79 (s, 1H), 8.60 (s, 1H), 8.39 (s, 1H), 7.85 (s,1H), 7.15 (s, 1H).

Example 7 4-Iodo-6-(pyridin-3-yl)pyrimidine (ACT-MIF-006)

The compound was prepared according to EXAMPLE 1 usingpyridine-3-boronic acid and 4,6-dichloropyrimidine. The resultant chlorocompound was converted to iodo with hydroiodic acid as described in thegeneral procedure.

¹H NMR (DMSO-d₆): δ 9.40 (s, 1H), 8.96 (s, 1H), 8.72 (m, 2H), 8.53 (m,1H), 7.52 (m, 1H).

Example 8 4-Iodo-6-(3-fluorophenyl)pyrimidine (ACT-MIF-008)

The compound was prepared according to EXAMPLE 1 using3-fluorophenylboronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 8.95 (s, 1H), 8.70 (s, 1H), 8.10 (m, 2H), 7.65 (m,1H), 7.45 (m, 1H).

Example 9 4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine (ACT-MIF-010)

The compound was prepared according to EXAMPLE 1 using4-tert-butyloxymethylphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (CDCl₃): δ 8.8 (s, 1H), 8.10 (s, 1H), 7.98 (m, 2H), 7.42 (m, 2H),4.71 (s, 2H), 1.50 (s, 9H).

Example 10 4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine (ACT-MIF-011)

The compound was prepared according to EXAMPLE 1 using2-fluoropyridine-3-boronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.94 (s, 1H), 8.70 (m, 1H), 8.40 (s, 1H), 7.69 (s,1H), 7.42 (m, 1H).

Example 11 4-Iodo-6-(furan-2-yl)pyrimidine (ACT-MIF-012)

The compound was prepared according to EXAMPLE 1 using furan-2-boronicacid and 4,6-dichloropyrimidine. The resultant chloro compound wasconverted to iodo with hydroiodic acid as described in the generalprocedure.

¹H NMR (DMSO-d₆): δ 8.80 (s, 1H), 8.22 (s, 1H), 8.01 (s, 1H), 7.50 (s,1H), 6.79 (s, 1H).

Example 12 4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine (ACT-MIF-013)

The compound was prepared according to EXAMPLE 1 using2-fluoropyridine-5-boronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (CDCl₃): δ 8.71 (s, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 8.15 (m,1H), 6.50 (m, 1H).

Example 13 4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine (ACT-MIF-014)

The compound was prepared according to EXAMPLE 1 using3-fluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (CDCl₃): δ 8.89 (s, 1H), 8.60 (s, 1H), 8.12 (m, 2H), 7.31 (m,1H), 3.92 (s, 3H).

Example 14 4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine (ACT-MIF-015)

The compound was prepared according to EXAMPLE 1 using2-chloropyridine-5-boronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 9.20 (m, 1H), 9.0 (s, 1H), 8.70 (s, 1H), 8.60 (m,1H), 7.72 (m, 1H).

Example 15 4-Iodo-6-(2-hydroxyphenyl)pyrimidine (ACT-MIF-016)

The compound was prepared according to EXAMPLE 1 using2-trifluoromethoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 12.70 (s, 1H), 8.31 (s, 1H), 7.80 (m, 1H), 7.55 (m,3H), 6.61 (s, 1H).

Example 16 4-Iodo-6-(2,4-difluorophenyl)pyrimidine (ACT-MIF-017)

The compound was prepared according to EXAMPLE 1 using2,4-difluorophenylboronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 9.01 (s, 1H), 8.39 (s, 1H), 7.80 (m, 1H), 7.41 (m,2H).

Example 17 4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine (ACT-MIF-018)

The compound was prepared according to EXAMPLE 1 using2-fluoro-6-methoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.89 (s, 1H), 8.15 (s, 1H), 7.49 (m, 1H), 7.0 (m,2H).

Example 18 4-Iodo-6-(2-chlorophenyl)pyrimidine (ACT-MIF-019)

The compound was prepared according to EXAMPLE 1 using2-chlorophenylboronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure. 1H NMR (DMSO-d₆): δ 9.0 (s, 1H), 8.31 (s, 1H),7.67 (m, 2H), 7.57 (m, 2H).

Example 19 4-Iodo-6-(3-acetylaminophenyl)pyrimidine (ACT-MIF-021)

The compound was prepared according to EXAMPLE 1 using3-acetylaminophenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 10.15 (s, 1H), 9.10 (s, 1H), 8.50 (s, 1H), 8.25 (s,1H), 7.90 (m, 2H), 7.55 (m, 1H), 2.10 (s, 3H).

Example 20 4-Iodo-6-(thiophen-3-yl)pyrimidine (ACT-MIF-022)

The compound was prepared according to EXAMPLE 1 usingthiophene-3-boronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 8.88 (s, 1H), 8.58 (s, 1H), 8.50 (s, 1H), 7.88 (m,1H), 7.71 (m, 1H).

Example 21 4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine (ACT-MIF-023)

The compound was prepared according to EXAMPLE 1 using3-tert-butyloxymethylphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.92 (s, 1H), 8.58 (s, 1H), 8.22 (m, 1H), 8.19 (m,1H), 7.50 (m, 2H), 4.60 (s, 2H).

Example 22 4-Iodo-6-(isoquinolin-4-yl)pyrimidine (ACT-MIF-025)

The compound was prepared according to EXAMPLE 1 usingisoquinoline-4-boronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 9.69 (s, 1H), 9.30 (m, 1H), 9.05 (s, 1H), 8.90 (s,1H), 8.15 (m, 2H), 7.90 (m, 1H), 7.70 (m, 1H).

Example 23 4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine (ACT-MIF-027)

The compound was prepared according to EXAMPLE 1 using2,4,5-trifluorophenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 9.0 (s, 1H), 8.32 (s, 1H), 8.12 (m, 1H), 7.81 (m,1H).

Example 24 4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine (ACT-MIF-028)

The compound was prepared according to EXAMPLE 1 using2,6-difluoropyridine-3-boronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 9.05 (s, 1H), 8.75 (m, 1H), 8.36 (s, 1H), 7.40 (m,1H).

Example 25 4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine (ACT-MIF-029)

The compound was prepared according to EXAMPLE 1 using2-methoxypyridine-5-boronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 9.10 (d, 1H), 8.90 (s, 1H), 8.61 (s, 1H), 8.45 (m,1H), 7.0 (m, 1H), 3.92 (s, 3H).

Example 26 4-Iodo-6-(thiophen-2-yl)pyrimidine (ACT-MIF-030)

The compound was prepared according to EXAMPLE 1. Specifically, thefollowing method was employed:

1. Preparation of 4-chloro-6-thiophen-2-yl-pyrimidine (3)

4,6-dichloropyrimidine (22.2 g, 149.1 mmol), thiophene-2-boronic acid(20.0 g, 156.3 mmol, 1.05 equiv), Na₂CO₃ (28.8 g, 271.4 mmol, 1.8 equiv)and Pd(PPh₃)₂Cl₂ (2.9 g, 4.2 mmol, 0.03 equiv) were refluxed indimethoxyethane-water (727:238 mL) mixed solvent system for 16 h.Reaction was monitored by TLC (using ethyl acetate:n-hexane, 1:9).Reaction mixture was cooled and the subject compound was extracted usingdichloromethane. Subject compound was purified by flash chromatography(5% ethyl acetate:n-hexane) to yield 18.4 g of 3 (Yield=62.8%).

¹H NMR (CDCl₃): 8.90 (d, J=0.9 Hz 1H), 7.79-7.80 (dd, J=3.9, 1.2 Hz,1H), 7.58-7.60 (m, 2H), 7.18-7.20 (m, 1H).

2. Preparation of 4-iodo-6-thiophen-2-yl-pyrimidine (4)

Aqueous solution of HI (63.5 g, 496.5 mol, 13.9 equiv) was charged to4-chloro-6-thiophen-2-yl-pyrimidine (3, 7.0 g, 35.6 mmol) and stirringwas continued for 20 h. Reaction mixture was then made slightly alkaline(pH ˜10) by using 5% NaOH solution. Subject compound was precipitatedout, filtered, washed well with distilled water and dried under vacuumto yield 9.6 g of 4 (Yield=94.1%).

HPLC=93.1%

To convert the unreacted chloro-, the product was again treated with HI(6.1 g, 47.7 mmol, 13.9 equiv) by following the same procedure asmentioned above to get 10.0 g of 4 (Yield=98.0%).

¹H NMR (CDCl₃): 8.76 (s, 1H), 8.02 (s, 1H), 7.76 (d, J=3.9 Hz, 1H), 7.58(d, J=4.8 Hz, 1H), 7.16-7.19 (m, 2H).

HPLC=99.12%

Example 27 4-Iodo-6-(3,4-difluorophenyl)pyrimidine (ACT-MIF-032)

The compound was prepared according to EXAMPLE 1 using3,4-difluorophenylboronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 8.94 (s, 1H), 8.69 (s, 1H), 8.31 (m, 1H), 8.13 (m,1H), 7.68 (m, 1H).

Example 28 4-Iodo-6-(4-ethoxyphenyl)pyrimidine (ACT-MIF-033)

The compound was prepared according to EXAMPLE 1 using3-fluoro-4-ethoxyphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.89 (s, 1H), 8.60 (s, 1H), 8.05 (m, 1H), 7.21 (m,2H), 4.20 (m, 2H), 1.32 (m, 3H).

Example 29 4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine (ACT-MIF-034)

The compound was prepared according to EXAMPLE 1 using4-aminocarbamoylphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.99 (s, 1H), 8.7 (s, 1H), 8.30 (m, 2H), 8.12 (s,1H), 8.0 (m, 2H), 7.51 (s, 1H).

Example 30 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine (ACT-MIF-035)

The compound was prepared according to EXAMPLE 1 using3-aminocarbamoylphenylboronic acid and 4,6-dichloropyrimidine. Theresultant chloro compound was converted to iodo with hydroiodic acid asdescribed in the general procedure.

¹H NMR (DMSO-d₆): δ 8.95 (s, 1H), 8.65 (m, 2H), 8.40 (m, 1H), 8.19 (s,1H), 8.08 (m, 1H), 7.62 (m, 2H).

Example 31 4-Iodo-6-(quinolin-4-yl)pyrimidine (ACT-MIF-036)

The compound was prepared according to EXAMPLE 1 usingquinoline-4-boronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 9.11 (s, 1H), 9.0 (s, 1H), 8.47 (s, 1H), 8.12 (m,2H), 7.81 (m, 1H), 7.7 (s, 1H), 7.61 (m, 1H).

Example 32 4-Iodo-6-(quinolin-8yl)pyrimidine

The compound was prepared according to EXAMPLE 1 usingquinolin-8-boronic acid and 4,6-dichloropyrimidine. The resultant chlorocompound was converted to iodo with hydroiodic acid as described in thegeneral procedure.

¹H NMR (DMSO-d₆): δ 8.80 (s, 1H), 8.49 (m, 1H), 8.30 (s, 1H), 7.98 (m,1H), 7.88 (s, 1H), 7.62 (m, 2H), 7.52 (m, 1H).

Example 33 4-Iodo-6-(quinolin-3-yl)pyrimidine

The compound was prepared according to EXAMPLE 1 usingquinolin-3-boronic acid and 4,6-dichloropyrimidine. The resultant chlorocompound was converted to iodo with hydroiodic acid as described in thegeneral procedure.

¹H NMR (DMSO-d₆): δ 9.65 (s, 1H), 9.23 (s, 1H), 9.01 (s, 1H), 8.87 (s,1H), 8.01 (m, 2H), 7.90 (m, 1H), 7.66 (m, 1H).

Example 34 4-Iodo-6-(isoquinolin-5-yl)pyrimidine

The compound was prepared according to EXAMPLE 1 usingisoquinolin-5-boronic acid and 4,6-dichloropyrimidine. The resultantchloro compound was converted to iodo with hydroiodic acid as describedin the general procedure.

¹H NMR (DMSO-d₆): δ 9.41 (s, 1H), 9.09 (s, 1H), 8.51 (m, 1H), 8.42 (s,1H), 8.30 (m, 1H), 8.10 (m, 2H), 7.80 (m, 1H).

Example 35 Methods for the Preparation of 2-alkylthio Derivatives

The compounds of Examples 36-39 are prepared using the method of EXAMPLE35.

Example 36 2-Methylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using methyl iodide asone of the reactants.

¹H NMR (CDCl₃): δ 8.03-8.06 (m, 2H), 7.82 (s, 1H), 7.49-7.54 (m, 3H),2.62 (s, 3H).

Example 37 2-Ethylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using ethyl iodide asone of the reactants.

¹H NMR (CDCl₃): δ 7.95-7.96 (m, 2H), 7.74 (s, 1H), 7.39-7.48 (m, 3H),3.14 (q, J=7.2 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

Example 38 2-Isopropylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using isopropyl iodideas one of the reactants.

¹H NMR (DMSO-d₆): δ 8.25 (s, 1H), 8.17-8.20 (m, 2H), 7.51-7.59 (m, 3H),3.89-3.99 (h, J=6.9 Hz, 1H), 1.42 (d, J=6.9 Hz, 6H).

Example 39 2-n-Butylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to Scheme-2 using n-butyl iodide asone of the reactant.

¹H NMR (DMSO-d₆): δ 8.26 (s, 1H), 8.18-8.20 (m, 2H), 7.54-7.59 (m, 3H),3.18 (t, J=7.2 Hz, 2H), 1.65-1.73 (m, J=7.2 Hz, 2H), 1.41-1.49 (m, J=7.2Hz, 2H), 0.93 (t, J=7.2 Hz, 3H).

Example 40 Methods for the Preparation of 2-Alkylamino Derivatives

The compounds of Examples 41-45 were prepared according to Scheme 3 ofEXAMPLE 40.

Example 41 2-Methylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using methylamine asRNH₂.

¹H NMR (CDCl₃): δ 7.98-8.00 (br s, 2H), 7.43-7.49 (m, 3H), 7.40 (s, 1H),5.24 (br s, 1H), 3.06 (d, J=3.0 Hz).

Example 42 2-Ethylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using ethylamine asRNH₂.

¹H NMR (CDCl₃): δ 7.97-7.99 (m, 2H), 7.44-7.48 (m, 3H), 7.39 (s, 1H),5.20 (br s, 1H), 3.48-3.57 (m, J=7.2 Hz, 1.2 Hz, 2H), 1.28 (t, J=7.2 Hz,3H).

Example 43 2-Propylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using propylamine asRNH₂.

¹H NMR (CDCl₃): δ 7.92 (br s, 2H), 7.35-7.44 (m, J=6.6 Hz, 3H), 7.31 (s,1H), 5.21 (br s, 1H), 3.38 (q, J=6.9 Hz, 2H), 1.53-1.65 (m, J=6.9, 7.3Hz, 2H), 0.93 (t, J=7.3 Hz, 3H).

Example 44 2-Isopropylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using isopropylamineas RNH₂.

¹H NMR (CDCl₃): δ 8.04-8.07 (m, 2H), 7.54-7.56 (m, 3H), 7.41 (s, 1H),6.98 (br s, 1H), 4.29-4.36 (m, J=6.9, 3.3 Hz, 1H), 1.34 (d, J=6.9, 6H).

Example 45 2-n-Butylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using n-butylamine asRNH₂.

¹H NMR (CDCl₃): δ 7.97 (br s, 2H), 7.45-7.48 (m, 3H), 7.38 (s, 1H), 5.30(br s, 1H), 1.57-1.64 (m, J=6.0 Hz, 2H), 1.40-1.47 (h, J=6.0, 2H), 0.96(t, J=6.0 Hz, 3H).

The compounds of Examples 46-56 are also prepared according the Scheme1.

Example 46 4-Iodo-6-(benzothiophen-2-yl)pyrimidine

Example 47 4-Iodo-6-(benzofuran-2-yl)pyrimidine

Example 48 4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine

Example 49 4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine

Example 50 4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine

Example 51 4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine

Example 52 4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine

Example 53 4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine

Example 54 4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine

Example 55 4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine

Example 56 4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine

Example 57 Solubility and Stability

Solubility of exemplary compounds in varying solvents is shown in Table2. The stability of the compounds in solution was examined by HPLCconcomitantly. Results indicated no degradation after 2 months stored atroom temperature.

TABLE 2 Solubilities of the Compounds of the Invention at 20-22° C.(mg/ml) MIF MIF MIF MIF MIF MIF 001 002 003 006 035 038 Ethanol 85.0 1.44.7 DMSO 216 — 150 Propane-diol 25 2.7 PEG-300 66 13.3 19.7 Corn oil 15<6 <6 Ethanol/Tween 20 10 10 80 Cremophor 15 14.5

Example 58 Cell Permeability and Transport

Cell permeability and transport mechanisms in Caco-2 and MDR1-MDCKmonolayers experiments were performed in triplicate in theapical-to-basolateral and basolateral-to-apical direction usingTRANSWELL® wells containing either Caco-2 or MDR1-MDCK monolayers. Amodified Hanks buffer pH 7.4 was used in both reservoir and receiverwells with the addition of 1% BSA in the receiver side. Confluentmonolayers were used and their integrity was verified using referencecompounds (Atenolol as a low permeability reference compound andPropanolol as a high permeability reference compound). A sample in thebasolateral and apical sides was taken after 2 hours and theconcentration measured by LC/MS-MS. Results are summarized in Table 3.The results also suggest that the compounds are not P-gp substrates andmay cross the blood brain barrier.

TABLE 3 Cell Permeability and Transport R(Caco-2) Caco-2 PermeabilityPapp (10⁴ cm/s) Permeability A-B B-A Efflux Class ACT-MIF-001 <0.1 <0.1— Low ACT-MIF-002 0.2 04 1.7 Low ACT-MIF-003 2.1 2.6 1.2 HighACT-MIF-006 5.4 7.8 1.4 High ACT-MIF-011 12.6 7.8 0.6 High ACT-MIF-0250.4 0.6 1.5 Low ACT-MIF-029 3.1 3.1 1.0 High ACT-MIF-033 2.7 3.0 1.1High ACT-MIF-035 3.1 3.3 1.1 High ACT-MIF-038 0.9 06 0.7 High CellPermeability and Transport Results (MDR1-MDCK) MDR1-MDCK PermeabilityPapp (10⁴ cm/s) P-gp A-B B-A Substrate Efflux Brain (1) ACT-MIF-001 2.31.9 No Low Low ACT-MIF-001 + 3.3 3.1 CSA ACT-MIF-002 1.0 0.7 No High LowACT-MIF-002 + 1.9 2.0 CSA ACT-MIF-003 3.3 3.3 No High High ACT-MIF-003 +5.2 4.7 CSA (1) Brain penetration classification (2)

Example 59 Microsomal Stability

Stability in human liver microsomes was tested over 24 hours at 37° C.using pooled mixed gender human liver microsomes. Liver microsomes wereprepared at 1.0 mg/ml of microsomal protein in a 100 mM potassiumphosphate pH 7.4 buffer with 1 mM NADPH. The media was incubated at 37°C. with the compound in solution in DMSO. The concentration of thecompound was followed by LC/MS-MS as a function of time. Samples wereassayed at t=0, 30, 60 and 120 minutes. Testosterone was used as apositive control. The same experiment was performed with mouse livermicrosomes instead of human liver microsomes. Results are summarized inTable 4.

TABLE 4 Metabolic Stability Determined from Stability in HumanMicrosomes Metabolic Stability in Human Microsomes % Remaining 0 min 15min 60 min ACT-MIF-001 100 <2 ACT-MIF-002 100 <2 ACT-MIF-003 100 57ACT-MIF-006 100 82 ACT-MIF-017 100 0 ACT-MIF-021 100 73 ACT-MIF-029 1006.4 ACT-MIF-033 100 82 ACT-MIF-035 100 92 ACT-MIF-038 100 47Testosterone 100 56

Example 60 Plasma Protein Binding

Plasma protein binding was ascertained using dialysis equilibriummethods known in the art. Results are summarized in Table 5. Warfarinwas used a high protein binding control.

TABLE 5 Human Plasma Protein Binding Human Plasma Protein Binding %Bound Compound Warfarin ACT-MIF-001 98.3 99.0 ACT-MIF-002 97.9 98.9ACT-MIF-003 96.2 98.9

Example 61 MIF Liver Lysates Enzymatic Activity

This experiment, using an ex-vivo approach and the tautomeric reactionof L-dopachrom, was designed to ascertain the level of inhibition of MIFfollowing administration of the compounds of this invention via oral,IV, IP or any other route of administration. Mice were used in theexample illustrated below, but other animals could be used as well.Groups of 3 mice were administered IP 1 mg of 4-IPP and ACT-002resuspended in 100 μl of corn oil every day for 3 days. Mice weresacrificed 6 hours after the last injection and livers were harvested.˜1 gram pieces of liver were lysed in PBS containing 1 mM NaVO₄, 2 mMNaF and a protease inhibitor cocktail (Roche Biochemical, Indianapolis,Ind.) using dounce-homogenization on ice. 500 μg of liver lysates wereadded to a final volume of 700 μl PBS in plastic cuvettes. 4 mML-3,4-dihydroxyphenylalanine methyl ester and 8 mM sodium periodate(Sigma-Aldrich) were combined in a 3:2 ratio to form L-dopachrome methylester. 300 μL of L-dopachrome methyl ester was then immediately added tothe cuvettes; the OD_(475 nm) was measured 2 min and 4 min afteraddition of the L-dopachrome. As shown in FIG. 1 (DMSO was used as anegative control—no inhibition of MIF), there is a significant in vivoinhibition of MIF indicating that the compound interacts with the MIFbinding pocket.

Example 62 MIF Tumor Lysate Enzymatic Activity

The ex vivo MIF enzymatic activity of tumor extracts/lysates followingin vivo dosing can be estimated in a manner similar to the method ofEXAMPLE 61. Tumor bearing mice were administered 1 mg/kg daily for 3days. 6 hours following the last dose, animals were sacrificed andtumors were resected and processed as described in EXAMPLE 61.Inhibition was also ascertained as in EXAMPLE 61. Results, shown in FIG.2, demonstrate significant inhibition of MIF in tumor lysates.

Example 63 Inhibition of Tumor Cells Proliferation

Inhibition of the proliferation of tumor cells was investigated in vitroin several tumor cell lines. Cells of the desired tumor cell line wereplated at 2×10⁵ cells/ml in 96 well plates. Twice the indicatedconcentrations of the compounds of the invention were added to cells thefollowing day in an equal volume of media. 72 hours later, cells werelysed and subjected to ATP determination using the CellTiterGlo-Luminescent Cell Viability Assay kit (Promega, Madison, Wis.).Experiments were done in triplicate. Results for the inhibition of cellsproliferation are reported as IC50 (the concentration leading to a 50%inhibition of proliferation of the cell population) and are listed inTable 6. FIGS. 3 and 4 show bar graphs comparing the IC50s of specificembodiments of compounds of the invention across multiple tumor celllines.

TABLE 6 IC50s for Compounds in Selected Tumor Cell Lines IC50 (microM)Du 145 ACT-MIF-001 <10 ACT-MIF-002 24.9 ACT-MIF-003 16.5 ACT-MIF-00636.7 ACT-MIF-017 <10 ACT-MIF-022 <40 ACT-MIF-029 <20 ACT-MIF-033 <5ACT-MIF-034 <100 ACT-MIF-035 21.7 ACT-MIF-038 9.2

Example 64 p53 Up Regulation

The up regulation of p53 was determined using a commercially availablep53 luciferase assay kit. 1×10⁵ cells/ml were plated in a 24 well plateand allowed to adhere overnight. MIF antagonists were added to the cellsat the indicated concentrations for 16 hours and transientlyco-transfected with 0.125 μg/well of p53-responsive luciferase promoterplasmid (Promega, Madison, Wis.) together with 0.0125 μg/well RenillapRL-null plasmid (Promega) using Lipofectamine (Invitrogen) transfectionreagent. After 24 hrs, Firefly and Renilla luciferase activities weremeasured by the Dual Luciferase in Reporter Assay System (Promega,Madison, Wis.) on a TD-20/20 luminometer (Turner Designs). Resultsrepresented in FIG. 5 indicate the compounds of the invention areimplicated in p53 regulation.

Example 65 MIF Cell Lysate Enzymatic Inhibition

Normal or transformed cell lysates can be used to determine theconcentration inhibiting the enzymatic activity of MIF present in celllysates. Cells are cultured in the appropriate media to the requirednumber of cells, collected, and lysed. Compounds to be characterized aresolubilized in DMSO and serial dilutions are performed in order toobtain a range of concentrations including complete and no quantifiableinhibition. Results, reported as IC50 (concentration leading to aninhibition of 50% of the MIF enzymatic activity), are summarized inTable 7.

TABLE 7 IC50 Values for MIF Cell Lysate Enzymatic Activity InhibitionIC50 (nM) 4-IPP >2000 ACT-MIF-001 37 ACT-MIF-002 70 ACT-MIF-003 200ACT-MIF-006 250 ACT-MIF-017 190 ACT-MIF-021 570 ACT-MIF-029 140ACT-MIF-033 115 ACT-MIF-034 270 ACT-MIF-035 185 ACT-MIF-036 230ACT-MIF-037 >1000 ACT-MIF-039 195

Example 66 Inhibition of Cell Migration and Invasion

The LOX-IMV1 tumor cell line was used to determine the inhibition ofcell migration using the Oris Cell Migration Assay kit (Promega, Mich.).Briefly, adherent cells were seeded into each well of the kit accordingto kit instructions. Concentrations of cells in the migration zone weredetermined to calculate IC50 values. Prior to the migration assay, cellproliferation IC50s were determined to differentiate between inhibitionof proliferation and migration. Results are shown in FIGS. 6 and 7.Results show a significant inhibition of migration even at very lowconcentration (0.03 μM). A slightly modified method was also used todetermine the inhibition of invasion. As shown in FIG. 7, invasion wasalso inhibited.

Example 67 Determination of the Anti-Angio Genic Properties in the ChickChorioallantoic Membrane (CAM) Assay

8 groups with 10 embryos in each group were used in the experimentdescribed below. Fresh fertile eggs were incubated for 3 days in astandard egg incubator at 37° C. for 3 days. On Day 3, eggs were crackedunder sterile conditions and embryos were placed into 20×100 mm plasticplates and cultivated at 37° C. in an embryo incubator with a waterreservoir on the bottom shelf. Air was continuously bubbled into thewater reservoir using a small pump so that the humidity in the incubatoris kept constant. On Day 6, a sterile silicon “o” ring was placed oneach CAM and test compound dissolved in 0.5% methylcellulose was placedinto each “o” ring in a sterile hood. Paclitaxel was used as a positivecontrol. Embryos were returned to the incubator after addition of testmaterial. Control embryos received 10 μL of vehicle alone. On Day 8,embryos were removed from the incubator and kept at room temperaturewhile blood vessel density were determined under each “o” ring using animage capturing system at a magnification of 160×. The blood vesseldensity was measured using an angiogenesis scoring system in thatarithmetic numbers 0 to 5 (or exponential numbers 1 to 32) are used toindicate number of blood vessels present at the treatment sites on theCAM. Number 5 represents the highest density and 0 represents noangiogenesis. The percent of inhibition at each dosing site wascalculated using the score recorded for that site divided by the meanscore obtained from the appropriate control samples for each individualexperiment. The percent of inhibition for each dose of a given compoundwas calculated by pooling all results obtained for that dose from 8-10embryos. Results are summarized in Table 8 below and demonstrate thatamong others, compounds ACT-MIF-001, ACT-MIF-002, and ACT-MIF-003 havehigh anti-angiogenic properties.

TABLE 8 Blood Vessel Densities Blood Vessel Density Conc per CAM — 6 nM0.3 nM 3 nM 30 nM Control 14.0 ± 3.2 Paclitaxel 2.8 ± 0.7 ACT-MIF-00110.5 ± 3.4  4.1 ± 3.4 1.8 ± 0.3 ACT-MIF-002 9.4 ± 2.4 8.6 ± 2.6 4.4 ±1.3 ACT-MIF-003 11.6 ± 1.2  4.2 ± 1.2 4.1 ± 0.7

Another experiment was performed using a protocol similar to the onedescribed above but using matrigel plugs instead of o ring to deliverthe test material to the CAM. Results are summarized in Table 9 belowand show a statistically significant inhibition of angiogenesis at thehigh concentrations of test material.

TABLE 9 Blood Vessel Densities Blood Vessel Counts Conc per CAM — 2 nM0.3 nM 3 nM 30 nM Control 39.3 ± 1.3 Paclitaxel 15.5 ± 2.1 ACT-MIF-00638.8 ± 3.5 35.8 ± 5.4 33.5 ± 1.7 ACT-MIF-030 35.6 ± 1.0 32.7 ± 3.2 28.1± 2.0 ACT-MIF-035 28.3 ± 1.7 27.3 ± 2.4 26.7 ± 1.9 ACT-MIF-038 31.8 ±5.8 33.6 ± 1.7 31.0 ± 4.1

Example 68 Pharmacokinetics Parameters

The pharmacokinetic parameters of several compounds were investigated inrodents. Both oral and iv administration were investigated in rats.Blood samples were collected over time; plasma was analyzed using anLC/MS-MS method. Pharmacokinetic parameters were calculated usingWin-NonLin. Terminal plasma half-lives were 7.10 hr for ACT-MIF-001,1.66 hr for ACT-MIF-002, and 1.50 hr for ACT-MIF-003. After i.v.administration, the clearance values were 45753 mL/hr/kg forACT-MIF-001, 7911 mL/hr/kg for ACT-MIF-002, and 11827 mL/hr/kg forACT-MIF-003. The volume of distribution values were 72666 mL/kg forACT-MIF-001, 2118 mL/kg for ACT-MIF-002, and 1926 mL/kg for ACT-MIF-003.

Example 69 In Vivo Efficacy in Xenograft Tumor Models

Athymic nude mice at 7-8 weeks of age were used for the study. Mice werehoused in microisolator housing, with food and water provided aslibitum, and quarantined for 4 days prior to the initiation of thestudy. DU145 cells were maintained in McCoy's 5A medium supplementedwith 10% fetal bovine serum and 2 mM glutamine. Cells at 80% confluencewere harvested using 0.25% trypsin/EDTA solution, washed once with PBSand resuspended in a mixture of serum-free medium/Matrigel (1:1 byvolume) at a density of 3×10⁶ cells/100 μl. 4 groups of 10 mice eachwere used in the experiment. DU145 cells suspended in 100 μl of amixture of medium/Matrigel (1:1) were subcutaneously implanted in theright flank region. Animals were monitored for tumor growth daily aftercell implantation. When tumor volumes reached 80-100 mm³, mice wererandomized into 4 groups of 10 mice each using only mice having tumorvolumes closest to the mean value. Tumor volumes were measured using theformula V=L×W×H×π/6, where L and W represent the longer and shorterdiameters of the tumor and H represents the height of the tumor.Treatment began the day after randomization. Act-MIF-001, ACT-MIF-002,and ACT-MIF-003 were administered daily by IP injection at a dose of 40mg/kg for 4 weeks. Throughout the entire study, tumor volumes weremeasured twice weekly and body weights once weekly. Animals wereobserved for possible toxic effect from the drug treatment. Resultsillustrated below in FIG. 8 demonstrated that ACT-MIF-003 significantlyinhibited tumor growth.

Example 70 Determination of the Microvessel Density in Xenograft Tumors

At the end of the experiment described in EXAMPLE 69 above, tumors ineach group were removed and sliced. Blood vessel density of the tumortissues was measured by immunohistochemistry. Results indicated adecrease in microvessel density with respect to the tumors of thecontrol group with a statistically meaningful difference for theACT-MIF-003 treated group. These in vivo results confirmed that thecompounds described in this application inhibit angiogenesis.Representative pictures of the stained tissues are showed in FIG. 9.

Example 71 Efficacy Study in a Pancreatic Tumor Model

The activity of the compounds of the invention was investigated in apancreatic tumor model using an experiment similar to the one describedin EXAMPLE 69. Compounds ACT-MIF-002 and ACT-MIF-003 were dosed daily at40 mg/kg via IP administration. Results shown in FIG. 10 indicated thatthe compounds of the invention tested in this experiment had asignificant impact on survival and that limited the metastatic tumorburden as shown in the survival graph and representative histopathologicslides (FIG. 11) of the lumbar region of control and treated animals. Inaddition, animal weights were monitored throughout the study; there wasno body weight loss and no clinical signs of toxicity indicating thatthese compounds are very well tolerated.

Lumbar regions of the control and treated groups were excised and sentfor histopathological evaluation. As shown in FIG. 11, there weresignificant differences between control and treated groups as there wasno evidence of bone metastases in the ACT-MIF-002 treated group. In theexample shown in FIG. 11, bone marrow of the vehicle treated mice isconsistent with bone metastases (1) with evidence of surroundingskeletal muscle metastases from invading marrow tumor cells (2 and 3).No evidence of bone metastases was observed with spinal column sectionsfrom ACT-MIF-002 treated mice.

Example 72 Oral Bioavailability

The compounds were administered orally (PO) and intraperitoneally (IP)to healthy animals. The inhibition of the MIF liver enzymatic activitydetermined ex vivo following IP and PO dosing is similar, indicatinghigh oral bioavailability. Furthermore, brain and lung tissues werecollected and processed to determine MIF enzymatic activity in theseorgans. Results also shown in FIG. 12 are indicative of an excellenttissue distribution and demonstrate significant MIF inhibition in boththe brain and lungs. As shown in FIG. 12, MIF-002, is orallybioavailable Inhibition of MIF enzyme was determined in vitro followingdosing of MIF-002 at 40 mg/kg once a day for three days, both IP and PO(normal C57BL6 mice, n=3). Tissues were collected at sacrifice andprocessed. Liver, lung, and brain tissues were collected, processed, andused for the determination of MIF enzyme activity. Values are expressedas a percentage calculated using DMSO as control (no inhibition).

Two additional compounds were tested, MIF-035 and MIF-041. Results (datanot shown) indicated that these compounds were also orally bioavailable,crossed the blood brain barrier, and inhibited MIF enzymatic activityvery efficiently in all three organs with results varying ˜12%inhibition in liver extracts to ˜76.2% inhibition in the lungs.

Results indicate compounds of the invention are orally bioavailable,cross the brain blood barrier, and inhibit MIF enzymatic activity inboth the brain and the lungs.

Example 73 4-IPP and ACT-003 Inhibit T Lymphocyte Activation

In order to assess the ability of MIF antagonists to disruptautoimmune-associated T cell activation, primary human T lymphocyteswere prepared using standard Ficoll-gradient preparations. 1×10⁶lymphocytes/ml were resuspended in RPMI/10% FCS and plated onto anti-CD3antibodies previously immobilized onto tissue culture plates. Control,vehicle control (0.1% DMSO), 25 μM 4-IPP or 25 μM ACT-003 were added tocells and allowed to incubate for 48 hours. Cells were lifted, washedand stained with anti-CD4 or anti-CD8 antibodies and then analyzed byflow cytometry. As shown in FIGS. 13 and 14, cells treated with MIFantagonists 4-IPP and ACT-003 during anti-CD3 lymphocyte activation hadsignificantly fewer CD4 and CD8 T lymphocytes suggesting defectiveanti-CD3 induced activation/proliferation in MIF inhibitor treatedlymphocytes.

To validate the effects of MIF antagonists on T lymphocyte activation,experiments were set up exactly as described above and, 48 hours later,treated and untreated lymphocytes were stained with an anti-CD25antibody. CD25 is also known as the high affinity IL-2 receptor—a verywell characterized and frequently marker of T lymphocyte activation. Asshown in FIGS. 15 and 16, 4-IPP and ACT-003 almost completely blockedthe anti-CD3-induced CD25 expression suggesting a nearly complete blockof T lymphocyte activation.

In order to investigate the relative kinetics of when 4-IPP and ACT-003are acting in blocking T lymphocyte activation, we repeated theexperiment described above but harvested lymphocytes only 16 hours afteranti-CD3 plating. At this early time point during T lymphocyteactivation, CD69 is found to be expressed and is usually considered tobe an “early marker” of lymphocyte activation. As shown in FIGS. 17 and18, treatment with MIF antagonists had only a marginal effect on CD69expression suggesting that MIF inhibitors are acting at a relativelylate stage in the activation process. This is important because itsuggests that therapeutic use of 4-IPP-based MIF inhibitors inautoimmune diseases can be used at later stages and aren't likely to berequired to be delivered in the early stages of disease onset.

Finally, to confirm that proliferation of CD4+ and CD8+ T lymphocytes isblocked by 4-IPP-based MIF antagonists, we repeated the experiment asdescribed above, added labeled-BrdU to cells, stained with eitherlabeled anti-CD4 or anti-CD8 antibodies and then assessed relativeCD4/CD8 and BrdU labeling in each treatment group. As shown in FIGS. 19and 20, 4-IPP and ACT-003 almost completely blocked BrdU labeling inboth CD8+ and CD4+ T lymphocytes.

Combined, these results suggest that targeting MIF using these4-IPP-based small molecules may have profound inhibitory effects on Tlymphocyte-dependent autoimmune disorders.

All documents cited are incorporated herein by reference; the citationof any document is not to be construed as an admission that it is priorart with respect to the present invention.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to one skilled in the artthat various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method for treating a macrophage migrationinhibitory factor (MIF)-implicated disease or condition, comprisingadministering to a patient in need thereof a safe and effective amountof a migration inhibitory factor (MIF) inhibitory compound or itsenantiomeric or diastereomeric form or a pharmaceutically acceptablesalt, prodrug, or metabolite thereof, said compound having the formula:

wherein: (i) A is selected from the group consisting of: (a) substitutedor unsubstituted 5, 6, or 7-membered aromatic or nonaromatic ringshaving 0 or 1 to 4 heteroatoms selected from the group consisting of N,O, S, and combinations thereof; (b) substituted or unsubstitutedbicyclic ring; (c) substituted or unsubstituted polycyclic rings; and(d) substituted or unsubstituted alkenes and alkynes having 2 to 10carbon atoms with 1 to 3 double or triple bonds; and  B is H, OH, OR,SR, NH₂, NHR, or alkyl or substituted alkyl, wherein R is H, alkyl, orsubstituted alkyl of 2 to 20 carbon atoms; or (ii) A is H or halo; and B is selected from the group consisting of: (a) substituted orunsubstituted 5, 6, or 7-membered aromatic or nonaromatic rings having 0or 1 to 4 heteroatoms selected from the group consisting of N, O, S, andcombinations thereof; (b) substituted or unsubstituted bicyclic ring;(c) substituted or unsubstituted polycyclic rings; and (d) substitutedor unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1to 3 double or triple bonds; and X and Y are independently N or CH,wherein at least one of X and Y is N.
 2. The method of claim 1, whereinthe MIF-implicated disease is selected from the group consisting ofinflammatory disease and cancer.
 3. The method of claim 2, wherein theinflammatory disease is selected from the group consisting ofdermatitis, arthritis, rheumatoid arthritis, insulin-dependent diabetes,proliferative vascular disease, acute respiratory distress syndrome,sepsis, septic shock, psoriasis, asthma, cytokine related toxicity,lupus, multiple sclerosis, transplant-host response, and autoimmunedisorders.
 4. The method of claim 1, wherein the MIF-implicatedcondition is caused by a MIF-producing pathogen.
 5. The method of claim4, wherein the MIF-producing pathogen is selected from the groupconsisting of parasitic helminths, spirochetes, and plasmodium.
 6. Themethod of claim 1, wherein the MIF inhibitory compound is selected fromthe group consisting of:4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-fluorophenyl)pyrimidine;4-Iodo-6-(4-fluorophenyl)pyrimidine; 4-Iodo-6-(furan-3-yl)pyrimidine;4-Iodo-6-(pyridin-3-yl)pyrimidine; 4-Iodo-6-(3-fluorophenyl)pyrimidine;4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(furan-2-yl)pyrimidine;4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;4-Iodo-6-(2-hydroxyphenyl)pyrimidine;4-Iodo-6-(2,4-difluorophenyl)pyrimidine;4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chlorophenyl)pyrimidine;4-Iodo-6-(3-acetylaminophenyl)pyrimidine;4-Iodo-6-(thiophen-3-yl)pyrimidine;4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;4-Iodo-6-(isoquinolin-4-yl)pyrimidine;4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;4-Iodo-6-(thiophen-2-yl)pyrimidine;4-Iodo-6-(3,4-difluorophenyl)pyrimidine;4-Iodo-6-(4-ethoxyphenyl)pyrimidine;4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;4-Iodo-6-(quinolin-4-yl)pyrimidine; 4-Iodo-6-(quinolin-8yl)pyrimidine;4-Iodo-6-(quinolin-3-yl)pyrimidine;4-Iodo-6-(isoquinolin-5-yl)pyrimidine;2-Methylthio-4-iodo-6-phenylpyrimidine;2-Ethylthio-4-iodo-6-phenylpyrimidine;2-Isopropylthio-4-iodo-6-phenylpyrimidine;2-n-Butylthio-4-iodo-6-phenylpyrimidine;2-Methylamino-4-iodo-6-phenylpyrimidine;2-Ethylamino-4-iodo-6-phenylpyrimidine;2-Propylamino-4-iodo-6-phenylpyrimidine;2-Isopropylamino-4-iodo-6-phenylpyrimidine;2-n-Butylamino-4-iodo-6-phenylpyrimidine;4-Iodo-6-(benzothiophen-2-yl)pyrimidine;4-Iodo-6-(benzofuran-2-yl)pyrimidine;4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.
 7. The method of claim 1,wherein: A is a substituted or unsubstituted bicyclic ring selected fromthe group consisting of a quinoline, an isoquinoline, a benzofuran, anda benzothiophene; B is H; X and Y are both N, and further wherein theMIF inhibitory compound interacts with a MIF polypeptide present in thepatient in need thereof to inhibit an enzymatic activity of the MIFpolypeptide.
 8. The method of claim 7, wherein the MIF inhibitorycompound is selected from the group consisting of:4-Iodo-6-(isoquinolin-4-yl)pyrimidine;4-Iodo-6-(quinolin-4-yl)pyrimidine; 4-Iodo-6-(quinolin-8-yl)pyrimidine;4-Iodo-6-(quinolin-3-yl)pyrimidine;4-Iodo-6-(isoquinolin-5-yl)pyrimidine;4-Iodo-6-(benzothiophen-2-yl)pyrimidine; and4-Iodo-6-(benzofuran-2-yl)pyrimidine.
 9. The method of claim 1, whereinthe administering is via oral administration, intravenousadministration, intraperitoneal administration, or a combinationthereof.
 10. A method for inhibiting proliferation, cell migration,metastasis, and/or invasion of a tumor cell, and/or angiogenesisassociated with the presence of the tumor cell, the method comprisingcontacting the tumor cell with an effective amount of a MIF inhibitorycompound or its enantiomeric or diastereomeric form or apharmaceutically acceptable salt, prodrug, or metabolite thereof, saidcompound having the formula:

wherein: (i) A is selected from the group consisting of: (a) substitutedor unsubstituted 5, 6, or 7-membered aromatic or nonaromatic ringshaving 0 or 1 to 4 heteroatoms selected from the group consisting of N,O, S, and combinations thereof; (b) substituted or unsubstitutedbicyclic ring; (c) substituted or unsubstituted polycyclic rings; and(d) substituted or unsubstituted alkenes and alkynes having 2 to 10carbon atoms with 1 to 3 double or triple bonds; and  B is H, OH, OR,SR, NH₂, NHR, or alkyl or substituted alkyl, wherein R is H, alkyl, orsubstituted alkyl of 2 to 20 carbon atoms; or (ii) A is H or halo; and B is selected from the group consisting of: (a) substituted orunsubstituted 5, 6, or 7-membered aromatic or nonaromatic rings having 0or 1 to 4 heteroatoms selected from the group consisting of N, O, S, andcombinations thereof; (b) substituted or unsubstituted bicyclic ring;(c) substituted or unsubstituted polycyclic rings; and (d) substitutedor unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1to 3 double or triple bonds; and X and Y are independently N or CH,wherein at least one of X and Y is N, whereby proliferation, cellmigration, metastasis, and/or invasion of the tumor cell and/orangiogenesis associated with the presence of the tumor cell isinhibited.
 11. The method of claim 10, wherein the MIF inhibitorycompound is selected from the group consisting of:4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-fluorophenyl)pyrimidine;4-Iodo-6-(4-fluorophenyl)pyrimidine; 4-Iodo-6-(furan-3-yl)pyrimidine;4-Iodo-6-(pyridin-3-yl)pyrimidine; 4-Iodo-6-(3-fluorophenyl)pyrimidine;4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(furan-2-yl)pyrimidine;4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;4-Iodo-6-(2-hydroxyphenyl)pyrimidine;4-Iodo-6-(2,4-difluorophenyl)pyrimidine;4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chlorophenyl)pyrimidine;4-Iodo-6-(3-acetylaminophenyl)pyrimidine;4-Iodo-6-(thiophen-3-yl)pyrimidine;4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;4-Iodo-6-(isoquinolin-4-yl)pyrimidine;4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;4-Iodo-6-(thiophen-2-yl)pyrimidine;4-Iodo-6-(3,4-difluorophenyl)pyrimidine;4-Iodo-6-(4-ethoxyphenyl)pyrimidine;4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;4-Iodo-6-(quinolin-4-yl)pyrimidine; 4-Iodo-6-(quinolin-8yl)pyrimidine;4-Iodo-6-(quinolin-3-yl)pyrimidine;4-Iodo-6-(isoquinolin-5-yl)pyrimidine;2-Methylthio-4-iodo-6-phenylpyrimidine;2-Ethylthio-4-iodo-6-phenylpyrimidine;2-Isopropylthio-4-iodo-6-phenylpyrimidine;2-n-Butylthio-4-iodo-6-phenylpyrimidine;2-Methylamino-4-iodo-6-phenylpyrimidine;2-Ethylamino-4-iodo-6-phenylpyrimidine;2-Propylamino-4-iodo-6-phenylpyrimidine;2-Isopropylamino-4-iodo-6-phenylpyrimidine;2-n-Butylamino-4-iodo-6-phenylpyrimidine;4-Iodo-6-(benzothiophen-2-yl)pyrimidine;4-Iodo-6-(benzofuran-2-yl)pyrimidine;4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.
 12. The method of claim 10,wherein: A is a substituted or unsubstituted bicyclic ring selected fromthe group consisting of a quinoline, an isoquinoline, a benzofuran, anda benzothiophene; B is H; X and Y are both N, and further wherein theMIF inhibitory compound interacts with a MIF polypeptide present in thepatient in need thereof to inhibit an enzymatic activity of the MIFpolypeptide.
 13. The method of claim 12, wherein the MIF inhibitorycompound is selected from the group consisting of:4-Iodo-6-(isoquinolin-4-yl)pyrimidine;4-Iodo-6-(quinolin-4-yl)pyrimidine; 4-Iodo-6-(quinolin-8-yl)pyrimidine;4-Iodo-6-(quinolin-3-yl)pyrimidine;4-Iodo-6-(isoquinolin-5-yl)pyrimidine;4-Iodo-6-(benzothiophen-2-yl)pyrimidine; and4-Iodo-6-(benzofuran-2-yl)pyrimidine.
 14. The method of claim 10,wherein the tumor cell is present within a subject and the contactingresults from administering the MIF inhibitory compound or itsenantiomeric or diastereomeric form, or the pharmaceutically acceptablesalt, prodrug, or metabolite thereof to the subject orally,intravenously, intraperitoneally, or a combination thereof.
 15. Themethod of claim 14, wherein the MIF inhibitory compound or itsenantiomeric or diastereomeric form, or the pharmaceutically acceptablesalt, prodrug, or metabolite thereof is administered as part of apharmaceutical composition comprising a safe and effective amount of theMIF inhibitory compound and one or more pharmaceutically acceptableexcipients.
 16. The method of claim 15, wherein the pharmaceuticalcomposition is pharmaceutically acceptable for use in a human.
 17. Amethod for inhibiting autoimmune-associated activation of a T cell, themethod comprising contacting a T cell subject to autoimmune-associatedactivation with an effective amount of a MIF inhibitory compound or itsenantiomeric or diastereomeric form or a pharmaceutically acceptablesalt, prodrug, or metabolite thereof of claim 1, wherebyautoimmune-associated activation of the T cell is inhibited.
 18. Themethod of claim 18, wherein the T cell subject to autoimmune-associatedactivation is present within a mammal.
 19. A compound selected from thegroup consisting of: 4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(4-fluorophenyl)pyrimidine; 4-Iodo-6-(furan-3-yl)pyrimidine;4-Iodo-6-(pyridin-3-yl)pyrimidine; 4-Iodo-6-(3-fluorophenyl)pyrimidine;4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(furan-2-yl)pyrimidine;4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;4-Iodo-6-(2-hydroxyphenyl)pyrimidine;4-Iodo-6-(2,4-difluorophenyl)pyrimidine;4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;4-Iodo-6-(2-chlorophenyl)pyrimidine;4-Iodo-6-(3-acetylaminophenyl)pyrimidine;4-Iodo-6-(thiophen-3-yl)pyrimidine;4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;4-Iodo-6-(thiophen-2-yl)pyrimidine;4-Iodo-6-(3,4-difluorophenyl)pyrimidine;4-Iodo-6-(4-ethoxyphenyl)pyrimidine;4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;2-Methylthio-4-iodo-6-phenylpyrimidine2-Ethylthio-4-iodo-6-phenylpyrimidine;2-Isopropylthio-4-iodo-6-phenylpyrimidine;2-n-Butylthio-4-iodo-6-phenylpyrimidine;2-Methylamino-4-iodo-6-phenylpyrimidine;2-Ethylamino-4-iodo-6-phenylpyrimidine;2-Propylamino-4-iodo-6-phenylpyrimidine;2-Isopropylamino-4-iodo-6-phenylpyrimidine;2-n-Butylamino-4-iodo-6-phenylpyrimidine;4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.